The focus of this proposal is to test prevailing models of the molecular mechanism of action of Selenoprotein N (SepN). In humans, mutations that cause complete loss of SepN and mutations that affect the function of the RyR1 protein, the core protein component of the skeletal muscle Ryanodine Receptor intracellular Calcium Release Channel (RyR-CRC), result in congenital myopathies with a similar spectrum of cellular defects. Our recently published work (Jurynec, M. J. et al. (2008). "Selenoprotein N is required for ryanodine receptor calcium release channel activity in human and zebrafish muscle." PNAS 105: 12485-90.) identified SepN as a factor necessary for normal calcium mobilization in vivo and necessary for normal RyR- CRC function measured in vitro. RyR-CRCs isolated from zebrafish embryos or human diseased muscle lacking SepN no longer responded in vitro to changes in the solution redox environment indicating SepN as an essential component of the RyR-CRC redox sensor. Consistent with these findings, SepN has sequence motifs indicative of an oxidoreductase. As we found SepN physically associated with the RyR-CRC in vivo, we proposed that i) SepN interacts directly with the Calcium Release Channel, ii) SepN functions as a substrate- specific oxidoreductase that helps regulate activity of the CRC, and iii) defects in CRC function account for SepN-related myopathies. An alternative model has been recently set forth in which the primary function of SepN is to maintain the overall redox state of the cell and only indirectly affects muscle function (Arbogast, S. et al. (2009). "Oxidative stress in SEPN1-related myopathy: From pathophysiology to treatment." Ann Neurol 65: 677-686.). Our goal is to understand the mechanism by which SepN mediates the regulation of channel function as an entrance toward a broader understanding of how RyR-CRC activity may be regulated during normal development, perturbed in disease processes, or potentiated by treatments. The goal of this R03 "small grant" proposal is to test elements of our working hypothesis as an essential first step toward a longer-term study aimed at identifying i) the specific interactions between SepN and the RyR-CRC and ii) the specific RyR-CRC target sites modified by SepN activity. Here we will determine: Aim #1) if the redox function of SepN is required for normal RyR-CRC function, and Aim #2) if the RyR-CRC is a direct target of the SepN redox reaction. Understanding the function of SepN will identify pathways or physiological states required for normal muscle development and function. More specifically, if our working model is correct, understanding SepN function will identify a key mechanism by which calcium mobilization can be regulated in multiple cell signaling and cell physiology contexts. PUBLIC HEALTH RELEVANCE: Mutations that completely block expression or function of Selenoprotein N (SepN) cause birth defects affecting muscle development and function. Mutations affecting the intracellular Calcium Release Channel cause similar birth defects. Our recent studies indicate that SepN that helps regulate Calcium Release Channel function, explaining why loss of either factor has a common effect. The focus of this proposal is to understand the mechanism by which SepN regulates channel function as an entrance toward a broader understanding of how Calcium Release Channel activity may be regulated during normal development, perturbed in disease processes, or potentiated by treatments.